published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Thesis (Ph. D.)--Columbia University, 1899. / "This study was awarded the Cartwright Prize for 1899 by the Alumni Association of the College of Physicians and Surgeons, Columbia University, New York." "From Archives of neurology and psychopathology, volume II, 1899. Published simultaneously in the Archives of neurology and psychopathology and the Journal of comparative neurology, pages 21 to 319 and plates I to VII of volume II of the Archives being severally identical with pages 157 to 455 and plates XIV to XX of vol. IX of the Journal." Includes bibliographical references (p. 279-288).

The effects of hypothermia on cultured mammalian (mouse) spinal cord neurons which had been subjected to a defined physical trauma (amputation of a primary dendrite 100μM from the perikaryon) were investigated.

During amniote embryogenesis, the segmented pattern characteristic of the vertebral column appears early during development through the sequential formation of multipotent structures called somites. Somites differentiate subsequently into dermomyotome (giving rise later to skin and skeletal muscles) and sclerotome (giving rise to vertebral bone structures and cartilage). In addition, sclerotomes subdivide following their rostro-caudal intrasegmental boundary into an axon growth-permissive region (anterior half) and an axon growth-repulsive region (posterior half). This binary system instructs motor and sensory axon navigation, as well as neural crest cell migration, to ensure that the peripheral nervous system develops without obstruction by the future cartilage and bones of the vertebral column. Repellent cues are expressed in posterior half-sclerotomes in order to exclude navigating axons from “no-go” areas and restrict their growth to specific exit points of the future vertebral column. Interestingly, similar repellent cues (e.g. Eph/Ephrins) are expressed in the adult central nervous system (CNS) and have been shown to control connectivity and plasticity throughout life. Following brain or spinal cord injury, these repellent molecules are upregulated by reactive astrocytes accumulating at the lesion site, and may impede axon regeneration in this region. In this dissertation, I am presenting the results of a differential gene expression analysis of anterior and posterior half-sclerotomes, based on RNA-sequencing data and using the chick embryo as a model organism. This study led to the identification of molecules, previously uncharacterized in this system, that may play a role in adhesive and mechanical properties of somites and in axon guidance and fasciculation. I focused on the functional analysis of one molecule of the posterior half-sclerotome, the extracellular matrix protein Fibulin-2. To look at its role in the segmentation of spinal axons, I used ectopic misexpression in a subset of segments based on somite electroporation. The width of spinal nerve bundle growth was restricted by Fibulin-2 overexpression in posterior and anterior half-sclerotomes, suggesting a role in sharpening/controlling the path of spinal axon growth. In addition, I showed that this could occur via an interaction with the axon growth repellent Semaphorin 3A. Then I looked at the expression of Fibulin-2 in two models of CNS injury: mouse cerebral cortical stab injury and rat dorsal crush spinal cord injury. In both cases, I observed an increase in Fibulin-2 protein level compared to control. I also used primary cultures of rat cortical astrocytes to show that the expression of Fibulin-2 after inflammatory cytokine-induced activation is increased. Finally, I studied a candidate axon growth repellent previously identified in the laboratory. I explored the hypothesis that this repellent molecule is an O-glycosylated, spliced variant form of a known protein. To characterize this repellent molecule, I used RNA-sequencing data from chick embryonic somites and 2D gel electrophoresis of an astrocytic cell line protein extract. Together, these results suggested that the developing vertebral column and the adult CNS share molecular features to control axon growth and plasticity. This type of study could lead to the characterization of molecular systems that regulate axon growth, and to the identification of novel therapeutic targets in brain or spinal cord injury.

Orientador: Sara Teresinha Olalla Saad / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-22T11:44:55Z (GMT). No. of bitstreams: 1 Ribeiro_ThiagoBorsoi_D.pdf: 4887111 bytes, checksum: 5ba29a0d383225adef5209fb35a27d81 (MD5) Previous issue date: 2013 / Resumo: A avulsão de raízes motoras, na interface do sistema nervoso central e periférico, já bem descrito na literatura, promove uma significativa perda sináptica com degeneração de cerca de 80% dos motoneurônios afetados. Não existem estratégias eficazes que propiciem uma reversão ou amenização deste quadro, mas alguns estudos já mostram que o passo fundamental é preservar os motoneurônios afetados. Pesquisas em diferentes áreas com células-tronco (CT) adultas estão sendo realizadas nos últimos anos e apresentam resultados promissores para a medicina regenerativa. Investigações recentes têm apontado para diferentes fontes de CT em tecidos adultos tais como de medula óssea, de sangue de cordão umbilical, tecido muscular, tecido nervoso, líquido amniótico entre outras. De modo geral, estas células apresentam como características principais a capacidade de proliferação e a diferenciação para outros tipos celulares. Entretanto, os principais problemas para o uso clínico das CT adultas são: i) pequena quantidade de células multipotentes, ii) o controle da diferenciação, iii) insuficiência no número de células viáveis e iiii) difícil obtenção. Como alternativa às dificuldades anteriormente citadas, o tecido adiposo tem sido foco de intensos estudos, pois este tecido possui rica fonte de células pluripotentes, além de apresentarem características positivas como fácil acesso ao tecido adiposo subcutâneo, obtenção em quantidade abundante e processo de isolamento celular relativamente simples. Apesar deste tecido apresentar organização complexa, é na fração celular do estroma vascular que se encontra uma rica população de células pluripotentes. Dados de literatura demonstram que as células mesenquimais derivadas de tecido adiposo (AT-MSC - Células mesenquimais de tecido adiposo), mediante incubação com meios de cultura variados, diferenciam-se em adipócitos, osteócitos, mioblastos, hepatócitos, células vasculares entre outras. A diferenciação de AT-MSC em células neuronais ainda é alvo de discussões e críticas na literatura, pois não há protocolos estabelecidos que induzam a diferenciação em células neuronais funcionais absolutas. Ainda, recentes estudos atribuem um potencial neuroprotetor e uma capacidade imunomodulatória à produção de fatores neurotróficos e a produção de fatores solúveis pelas AT-MSCs, constituindo talvez o principal mecanismo de ação destas células in vivo. O xenotransplante também é alvo de criticas de diversos trabalhos. Células humanas em modelo animal têm sido usadas constantemente em diversos trabalhos e o uso ou não de imunossupressores é um dos motivos de questionamento dos resultados, uma vez que sistemas imunológicos diferentes podem causar reações imunológicas não pretendidas nos estudos e, consequentemente, os resultados não seriam confiáveis. Entretanto, artigos de revisões x bibliográficas demonstram que diversos estudos envolvendo células-tronco mesenquimais e modelos animais têm resultados promissores e corroboram entre si. Com objetivo de investigar a ação de células-troncos de tecido adiposo de lipoaspirado humano em modelo animal, o presente trabalho propôs analisar a capacidade de sobrevivência das AT-MSCs humanas no modelo de avulsão da raiz ventral de ratos, bem como sua capacidade de neuroproteção dos motoneurônios lesionados e sua competência de imunossupressão no período de 2 semanas pós lesão (período agudo). Análise da dinâmica das sinapses, da reação astroglial e microglial e da reação de linfócitos T tiveram o objetivo de identificar condições que promovessem a sobrevivência e regeneração dos motoneurônios axotomizados assim como a capacidade de atuação das células humanas em ratos. Deste modo, foi observado que o tratamento com as AT-MSC humanas tiveram efeito neuroprotetor, uma vez que houve aumento significativo de sobrevivência neuronal e promoveram a estabilidade sináptica. As células humanas também tiveram ação imunomodulatória, reduzindo a astrogliose reativa e ativação microglial, bem como inibiu a atividade de linfócitos T. Pode-se dizer que no modelo a ação das células-tronco mesenquimais humanas ocorre semelhante ao de transplantes alogênicos em modelos animais / Abstract: It is well described in the literature that avulsion motor at the interface of the central and peripheral nervous system, promotes a significant loss of synaptic degeneration and 80% of motor neurons death. There is no effective strategies that favor a reversal or mitigation of this framework, but some studies have shown that the key step is to preserve motor neurons affected. Researches in different areas with stem cell (CT) adults are being undertaken in recent years and show promising results for regenerative medicine. Recent investigations have pointed to different sources of CT in adult tissues such as bone marrow, umbilical cord blood, brain, muscle tissue, amniotic fluid, among others. Generally, these cells have as main characteristics capacity for proliferation and differentiation to other cell types. However, the main problems for the clinical use of adult SC are: i) small amount of multipotent cells, ii) differentiation control, iii) low number of viable cells and iiii) difficulty to obtain. As an alternative to the difficulties mentioned above, adipose tissue has been the focus of intense study, because this tissue has a rich source of stem cells, in addition to having positive characteristics such as easy access to subcutaneous adipose tissue, obtained in abundant quantities and isolation process relatively simple. Despite the complex tissue organization, the stromal vascular fraction is rich of pluripotent population cells. Literature data show that stromal cells derived from adipose tissue (AT-MSC-adipose tissue mesenchymal stem cells) can differentiate by incubation with various culture media into adipocytes, osteocytes, myoblasts, hepatocytes, vascular cells, among others. The AT-MSC differentiation into neuronal cells is still subject of discussion and criticism in literature, since no established protocol has induced differentiation into function neuronal cells. Still, many studies attribute a potential neuroprotective and immunomodulatory capacity for the production's AT-MSC of neurotrophic factors and the soluble factors, constituting perhaps the main mechanism of action of these cells in vivo. Xenotransplantation is the target of criticism of many studies. Human cells constantly used in animal models have been used in several works and the use of immunosuppressants or not is subject to questioning of the results, since different immune systems can cause unwanted immune reactions in studies and, consequently, the results would not be reliable. Literature reviews show that several studies involving mesenchymal stem cells and animal models have shown promising results and corroborate each other. xii To investigate the action of stem cells from human adipose tissue lipoaspirate animal model, this study proposes to analyse the survivability of the human AT-MSC in ventral root avulsion model in rats as well as its ability to neuroprotection in moto neurons and their competence in the period of immunosuppression 2 weeks post injury (acute period). To identify conditions that promote the survival and regeneration of axotomized motoneurons as well as the capacity for action of human cells in mice, synapses dynamic analysis, astroglial and microglial reaction and reaction of T lymphocytes was examined. Thus, it was observed that treatment with AT-MSC had neuroprotective effect, since there was a significant increase in neuronal survival and the AT-MSC human promoted synaptic stability. Human cells also have immunomodulatory action, reducing reactive astrogliosis and microglial activation and inhibited the activity of T lymphocytes. It is possible that the model action of human mesenchymal stem cells is similar to that of allogeneic / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutor em Fisiopatologia Medica

Objetivou-se estudar a origem e distribuição do plexo lombossacral de Sus scrofa. Foram utilizados 30 exemplares, sendo 13 fêmeas e 17 machos, após serem fixados em solução aquosa de formaldeído a 10%. Os javalis possuem cinco ou seis vértebras lombares e quatro sacrais. O plexo lombossacral origina-se dos segmentos medulares de L2 a S4. O nervo cutâneo femoral lateral de L2 a L4 ramifica-se nos músculos psoas maior e menor, oblíquo interno do abdome, linfonodos subilíacos e pele da região da articulação do joelho. O nervo femoral de L3 a L6 supre os músculos psoas maior, psoas menor, ilíaco, quadríceps femoral, pectíneo, emite nervo safeno que inerva o músculo sartório e fáscia e pele das articulações do joelho e tarso. O nervo obturatório de L3 a L6 distribui-se para os músculos obturador externo, pectíneo, grácil e adutor. Nervo glúteo cranial de L4 a S1 inerva os músculos glúteo médio e piriforme. Nervo glúteo caudal de L6 a S2 emite ramos para o músculo glúteo superficial. Nervo isquiático de L4 a S2 inerva os músculos tensor da fáscia lata, glúteo médio, glúteo profundo, glúteo acessório, gêmeos, quadrado femoral, semitendíneo, semimbranáceo, bíceps femoral. O nervo tibial supre os músculos gastrocnêmico, sóleo, poplíteo e flexores profundos dos dedos na região plantar divide-se em nervos plantares medial e lateral, os quais ramificam em digitais plantares. O nervo fibular comum divide-se em nervos fibular superficial e fibular profundo. O fibular profundo emite ramos para os músculos tibial cranial, fibular terceiro, extensor longos dos dedos, fibular longo, extensor lateral dos dedos e interósseo. O nervo fibular superficial supre a fáscia da articulação do tarso continua como nervos digitais dorsais. O nervo pudendo de S2 a S4 distribui-se para músculos, esfíncter externos do ânus, levantador do ânus, constritor da vulva, isquiocavernoso e bulboesponjoso, emite os ramos dorsal do clitóris e mamários que inervam clitóris e pele da região vulvar respectivamente, o ramo dorsal do pênis distribui-se na glande pênis, prepúcio e escroto. O nervo retal caudal de S2 a S4 distribui-se para os músculos levantadores do ânus, coccígeo e esfíncter externo do ânus. A formação do plexo lombossacral, assim como origens e distribuições dos nervos que o compõem são similares aos descritos na literatura alusiva aos animais domésticos. / It aimed to study the origin and distribution of the lumbosacral plexus of Sus scrofa. 30 specimens were used, with 13 females and 17 males, after being fixed in an aqueous 10% formaldehyde. Boars have five or six lumbar vertebrae and four sacral vertebrae. The lumbosacral plexus originates from the spinal cord segments from L2 to S4. The lateral femoral cutaneous nerve from L2 to L4 branches off to the psoas major and minor muscles, internal oblique of the abdomen, sub-iliac lymph nodes and the knee joint region skin. The femoral nerve L3 to L6 supplies the major psoas muscle, iliac minor psoas, femoral quadriceps, pectineus, emits saphenous nerve that innervates the sartorius muscle and fascia and skin of the knee and tarsus joints. The obturator nerve from L3 to L6 is distributed to the external obturator muscles, pectineus, gracilis and adductor. The gluteal cranial nerve from L4 to S1 innervates the gluteus medius and piriformis muscles. The gluteal caudal nerve from L6 to S2 emits branches to the superficial gluteal muscle. The sciatic nerve from L4 to S2 innervates the tensor muscles of the fascia lata, gluteus medius, deep gluteus, gluteus accessory, twins, femoral square, semitendinosus, semimembranosus, biceps femoris. The tibial nerve supplies the gastrocnemius, soleus, popliteal and the deep finger flexors in the plantar region that is divided into medial and lateral plantar nerves which branch off into digital plantar. The common fibular nerve is divided into superficial nerves and deep fibular. The deep fibular nerve emits branches to the cranial tibial, third fibular, long extensor of fingers, peroneus longus, lateral extensor digitorum and interosseous muscles. The superficial fibular nerve supplies the fascia joint of the tarsos, which continues as dorsal digital nerves. The pudendal nerve from S2 to S4 is distributed to muscles, external anal sphincter, levator ani, constrictor of the vulva, ischiocavernosus and bulbospongiosus, emits the dorsal branches of the clitoris and of the breast, that innervate the clitoris and skin of the vulvar region, respectively. The dorsal branch of the penis is distributed in the glans penis, prepuce and scrotum. The rectal caudal nerve from S2 to S4 is distributed to the levator ani, coccyx and external anal sphincter. The formation of the lumbosacral plexus, as well as sources and distributions of the nerves that form it, are similar to those described in the literature alluding to domestic animals. / Tese (Doutorado)

Networks of interneurons within the spinal cord coordinate the rhythmic activation of muscles during locomotion. These networks are subject to extensive neuromodulation, ensuring appropriate behavioural output. Astrocytes are proposed to detect neuronal activity via Gαq-linked G-protein coupled receptors and to secrete neuromodulators in response. However, there is currently a paucity of evidence that astrocytic information processing of this kind is important in behaviour. Here, it is shown that protease-activated receptor-1 (PAR1), a Gαq-linked receptor, is preferentially expressed by glia in the spinal cords of postnatal mice. During ongoing locomotor-related network activity in isolated spinal cords, PAR1 activation stimulates release of adenosine triphosphate (ATP), which is hydrolysed to adenosine extracellularly. Adenosine then activates A1 receptors to reduce the frequency of locomotor-related bursting recorded from ventral roots. This entails inhibition of D1 dopamine receptors, activation of which enhances burst frequency. The effect of A1 blockade scales with network activity, consistent with activity-dependent production of adenosine by glia. Astrocytes also regulate activity by controlling the availability of D-serine or glycine, both of which act as co-agonists of glutamate at N-methyl-D-aspartate receptors (NMDARs). The importance of NMDAR regulation for locomotor-related activity is demonstrated by blockade of NMDARs, which reduces burst frequency and amplitude. Bath-applied D-serine increases the frequency of locomotor-related bursting but not intense synchronous bursting produced by blockade of inhibitory transmission, implying activity-dependent regulation of co-agonist availability. Depletion of endogenous D-serine increases the frequency of locomotor-related but not synchronous bursting, indicating that D-serine is required at a subset of NMDARs expressed by inhibitory interneurons. Blockade of the astrocytic glycine transporter GlyT1 increases the frequency of locomotor-related activity, but application of glycine has no effect, indicating that GlyT1 regulates glycine at excitatory synapses. These results indicate that glia play an important role in regulating the output of spinal locomotor networks.

Indiana University-Purdue University Indianapolis (IUPUI) / Although peripheral motoneurons are phenotypically endowed with robust regenerative capacity, functional recovery is often suboptimal following peripheral nerve injury (PNI). Research to date indicates that the greatest success in achieving full functional recovery will require the use of a combinatorial approach that can simultaneously target different aspects of the post-injury response. In general, the concept of a combinatorial approach to neural repair has been established in the scientific literature but has yet to be successfully applied in the clinical situation. Emerging evidence from animal studies supports the use of electrical stimulation (ES) and testosterone as one type of combinatorial treatment after crush injury to the facial nerve (CN VII). With the facial nerve injury model, we have previously demonstrated that ES and testosterone target different stages of the regeneration process and enhance functional recovery after facial nerve crush injury. What is currently unknown, but critical to determine, is the impact of a combinatorial treatment strategy of ES and testosterone on functional recovery after crush injury to the sciatic nerve, a mixed sensory and motor spinal nerve which is one of the most serious PNI clinical problems. The results of the present study indicate that either treatment alone or in combination positively impact motor recovery. With regard to molecular effects,single and combinatorial treatments differentially alter the expression of regeneration-associated genes following sciatic nerve crush injury relative to facial nerve injury. Thus, our data indicate that not all injuries equally respond to treatment. Furthermore, the results support the importance of treatment strategy development in an injury-dependent manner and based upon the functional characteristics of spinal vs. cranial nerves.